G-protein-coupled receptors (GPCRs) are important ubiquitous regulators of cellular functions. Recently, the CIRL family of cell membrane receptors with unusual structural features was discovered. The CIRLs contain seven transmembrane hydrophobic segments, a hallmark of GPCRs, and are significantly homologous to the members of the secretin receptor family of GPCRs. However, in contrast with other GPCRs, they are large (about 200 kDa) and consist of two non-covalently bound heterologous subunits. These subunits result from proteolytic processing of a precursor polypeptide in its extracellular N-terminal region very close to the transmembrane core. The peripheral extracellular subunits of the CIRLs contain structural domains typical for cell adhesion proteins whereas the integral membrane subunits resemble generic GPCR. CIRL-1 and CIRL-2 bind alpha-latrotoxin, a neurotoxin which stimulates secretion from neuronal and endocrine cells. When over-expressed in chromaffin cells, these receptors inhibit the ATP-dependent stage of secretion. On the basis of the available structural and functional data, we hypothesize that the CIRL receptors are regulators of secretion in various types of cells that may be activated by cell-cell interactions. The long-term goal of our studies is to understand the signaling pathways which involve the CIRLs and their physiological importance. The specific aims of the proposed experiments are: 1. To determine the amino acid sequences of members of the CIRL family. In the preliminary studies, cDNAs encoding two novel members of this family in addition to originally described CIRL were identified and molecularly cloned. The available clones will be sequenced completely and the structure of different splice variants of CIRLs will be analyzed. Plasmid constructs containing full-length cDNAs will be prepared for expression of CIRLs in eucaryotic cells. 2. To analyze tissue and cell distribution of CIRLs. Northern blotting indicated that the members of the CIRL family have differential tissue distribution. CIRL-1 and CIRL-3 are preferentially expressed in brain tissues whereas CIRL-2 is ubiquitous with highest concentrations found in heart, placenta, kidney, pancreas, spleen, prostate and ovary. The distribution of CIRLs will be further analyzed in detail by in situ hybridization with specific probes in adult and developing rat tissues. 3. To test the functional properties of CIRLs in secretion assays. CIRL-2 and CIRL-3 will be expressed in chromaffin cells and in PC12 cells which allow to analyze coupling of receptors to regulated secretion. 4. To analyze the role of endogenous Proteolytic processing of CIRLs for their correct expression and function.